The present invention relates generally to isolated and purified proteins and nucleic acids which modulate stem cell renewal, growth and division and which modulate primordial germ cell proliferation. More particularly, the present invention relates to isolated and purified piwi family proteins and isolated and purified polynucleic acids encoding the same.
The publications and other materials used herein to illuminate the background of the invention, and in particular cases, to provide additional details respecting the practice, are incorporated herein by reference, and for convenience, are referenced by author and year of publication in the following text, and respectively grouped by author in the appended list of references.
Stem cells are a very small number of founder cells that play a central role in tissue development and maintenance. In human bodies, stem cells are responsible for generating and/or maintaining approximately 90% of cells in the adult tissues. Over-proliferation of malignant stem cells is the leading cause of cancer while under-proliferation of stem cells or stem-like progenitor cells leads to tissue dystrophy, anemia, immunodeficiency, and male infertility. The crucial role of stem cells has long been attributed to their ability to self-renew and to generate immense number of specialized cells on demand.
The ability of stem cells to self-renew and to produce a large number of differentiated progeny is critical for the development and maintenance of a wide variety of tissues in organisms ranging from insects to mammals (reviewed in Potten, 1997; Lin, 1997; Lin and Schagat, 1997; Morrison et al., 1997). This self-renewing ability is controlled both by extrinsic signaling and by cell-autonomous mechanisms (reviewed in Morrison et al., 1997; Lin and Schagat, 1997). Cell autonomous mechanisms have been elucidated in a few stem cell models such as neuroblasts and germline stem cells in Drosophila (Lin and Schagat, 1997; Deng and Lin, 1997), whereas the role of extrinsic signaling has been elucidated in several systems. For example, the proliferation and differentiation of mammalian stem cells in the hematopoietic, epidermal, and nervous systems depend on extrinsic signals that act on specific receptors on the stem cell surface (Morrison et al., 1997).
In diverse organisms ranging from invertebrates to mammals, the proliferation of germ cells, some of which possess stem cell properties, has been postulated, and, in some cases, shown to be regulated by neighboring non-mitotic somatic cells (Lin, 1997). Particularly, in C. elegans, cellxe2x80x94cell interactions between the somatic distal tip cell (DTC) at the end of each gonadal arm and the underlying mitotic germline nuclei via the lag-2lg/p-1 signaling pathway provides a paradigm for soma-germline interaction (reviewed in Kimble and Simpson, 1997). The glp-1 pathway is required to maintain a population of mitotically active nuclei in the germline.
However, few molecules and/or mechanisms identified in a particular type of stem cells have been shown to be applicable to other stem cell systems. For example, the glp-1 equivalent pathway in Drosophila does not play a role in regulating GSC division and maintenance (Ruohala et al., 1991; Xu et al., 1992).
The self-renewing asymmetric division of GSCs in the Drosophila ovary is known to be controlled both by an intracellular mechanism (Deng and Lin, 1997) and by cellxe2x80x94cell interactions (Lin and Spradling, 1993). The intracellular mechanism involves a cytoplasmic organelle termed the spectrosome that controls the orientation of GSC division (Lin et al., 1994; Deng and Lin, 1997). The cellxe2x80x94cell interaction mechanism involves terminal filament cells, as shown by laser ablation studies (Lin and Spradling, 1993). Recently, dpp has been shown as a key signaling molecule required for GSC division and maintenance (Xie and Spradling, 1998). It is possible that the dpp signal emanates from somatic cells. Alternatively, dpp signal may originate from the germline or even within GSCs, like its mammalian homologs (Zhao et al., 1996).
In mammals, primordial germ cells cultured from the genital ridge have the ability to give rise to pluripotent embryonic stem cells. For example, U.S. Pat. No. 5,690,926 issued Nov. 25, 1997 to Hogan; U.S. Pat. No. 5,670,372 issued Sep. 23, 1997 to Hogan; and U.S. Pat. No. 5,537,357 issued Sep. 26, 1995 to Hogan each disclose pluripotential mammalian embryonic stem cells and methods of making the same. The disclosure of these patents is limited to mammalian embryonic stem cells and particularly to the culturing of murine and other mammalian embryonic stem cells using a combination of growth factors consisting of SCF, FGF and LIF.
Current prior art reports on the culture of avian primordial germ cells (PGCs) have concentrated on efforts to maintain a PGC-phenotype and to stimulate proliferation. See e.g., Chang, I. K. et al., Cell. Biol. Int. 1997 August; 21(8): 495-9; Chang, I. K. et al., Cell. Biol. Int. 1995 February; 19(2): 143-9; Allioli, N. et al., Dev. Biol. 1994 September; 165(1): 30-7 and PCT Publication No. WO 99/06533, published Feb. 11, 1999 (Applicantxe2x80x94University of Massachusetts; Inventorsxe2x80x94Ponce de Leon et al.).
As illustrated above, numerous attempts have been devoted to identify genes that control the self-renewing ability of stem cells or the proliferation of primordial germ cells. As a result, a number of growth factors and signaling molecules, such as Steel factor and its c-kit receptor, have been identified to regulate such activity in certain tissues. Despite this progress, there remains a long-felt and continuing need to identify genes that play a role in modulating the growth and self-renewing division of stem cells, particularly GSCs, and that play a role in modulating proliferation of primordial germ cells.
The present invention contemplates an isolated and purified family of genes and gene products (the piwi family) which plays a role in the growth, proliferation and self-renewing division of stem cells, and proliferation of primordial germ cells. More preferably, a polypeptide of the invention is a recombinant polypeptide. Even more preferably, a polypeptide of the present invention comprises a vertebrate piwi family polypeptide. Even more preferably, a polypeptide of the present invention comprises a mammalian piwi family polypeptide. Even more preferably, a polypeptide of the present invention comprises a human piwi family polypeptide. Even more preferably, a polypeptide of the present invention comprises an amino acid sequence from the amino acid residue sequences of any of SEQ ID NOs:2, 4 and 6.
The present invention also provides an isolated and purified polynucleotide that encodes a polypeptide that plays a role in the growth, proliferation and self-renewing division of stem cells, and proliferation of primordial germ cells. In a preferred embodiment, a polynucleotide of the present invention comprises a DNA molecule from a vertebrate species. Preferred vertebrates comprise mammals, birds orfish. A preferred mammal is a human. More preferably, a polynucleotide of the present invention encodes a polypeptide designated PIWI. Even more preferably, a polynucleotide of the present invention encodes a polypeptide comprising an amino acid residue sequence of any of SEQ ID NOs:2, 4 and 6. Most preferably, an isolated and purified polynucleotide of the invention comprises a nucleotide base sequence of any of SEQ ID NOs:1, 3 and 5.
In another embodiment, the present invention contemplates an antibody immunoreactive with a piwi family polypeptide as described above. SEQ ID NOs:3-6 set forth nucleotide and amino acid sequences from representative vertebrates, human and mouse. Also contemplated by the present invention are antibodies immunoreactive with homologues or biologically equivalent piwi family polynucleotides and polypeptides found in other vertebrates. Preferably, an antibody of the invention is a monoclonal antibody. More preferably, the piwi family polypeptide comprises human PIWI (HIWI). Even more preferably, the piwi polypeptide comprises an amino acid residue sequence of any of SEQ ID NOs:2, 4 and 6.
In another aspect, the present invention contemplates a method of producing an antibody immunoreactive with a piwi family polypeptide as described above, the method comprising the steps of (a) transfecting a recombinant host cell with a polynucleotide that encodes a biologically active piwi family polypeptide; (b) culturing the host cell under conditions sufficient for expression of the polypeptide; (c) recovering the polypeptide; and (d) preparing the antibody to the polypeptide. SEQ ID NOs:3-6 set forth nucleotide and amino acid sequences from representative vertebrates, human and mouse. Preferably, the host cell is transfected with a polynucleotide of any of SEQ ID NOs:1, 3 and 5. Even more preferably, the present invention provides an antibody prepared according to the method described above. Also contemplated by the present invention is the use of homologues or biologically equivalent polynucleotides and polypeptides found in other vertebrates to produce antibodies.
Alternatively, the present invention provides a method of detecting a piwi family polypeptide as described above, wherein the method comprises immunoreacting the polypeptide with an antibody prepared according to the method described above to form an antibody-polypeptide conjugate, and detecting the conjugate.
In yet another embodiment, the present invention contemplates a method of detecting a messenger RNA transcript that encodes a piwi family polypeptide as described above, wherein the method comprises hybridizing the messenger RNA transcript with a polynucleotide sequence that encodes that polypeptide to form a duplex; and detecting the duplex. Alternatively, the present invention provides a method of detecting a DNA molecule that encodes a piwi family polypeptide as described above, wherein the method comprises hybridizing DNA molecules with a polynucleotide that encodes a biologically active piwi family polypeptide to form a duplex; and detecting the duplex.
In another aspect, the present invention contemplates an assay kit for detecting the presence of a piwi family polypeptide in a biological sample, where the kit comprises a first container containing a first antibody capable of immunoreacting with a biologically active piwi polypeptide, with the first antibody. Preferably, the first antibody is present in an amount sufficient to perform at least one assay. Also preferably, an assay kit of the invention further comprises a second container containing a second antibody that immunoreacts with the first antibody. More preferably, the antibodies used in an assay kit of the present invention are monoclonal antibodies. Even more preferably, the first antibody is affixed to a solid support. More preferably still, the first and second antibodies comprise an indicator, and, preferably, the indicator is a radioactive label, a fluorescent label or an enzyme.
In an alternative aspect, the present invention provides an assay kit for detecting the presence, in biological samples, of a piwi family polypeptide, the kits comprising a first container that contains a second polynucleotide identical or complementary to a segment of at least 10 contiguous nucleotide bases of a polynucleotide that encodes a biologically active piwi family polypeptide.
In another embodiment, the present invention contemplates an assay kit for detecting the presence, in a biological sample, of an antibody immunoreactive with a piwi family polypeptide, the kit comprising a first container containing a biologically active piwi family polypeptide that immunoreacts with the antibody, with the polypeptide present in an amount sufficient to perform at least one assay.
In still a further embodiment, this invention pertains to transgenic, therapeutic, screening and culturing methods based upon the self-renewing division of stem cells, and proliferation of primordial germ cells function of piwi family polynucleotides and polypeptides as described herein. Representative therapeutic and culturing methods include administration of a soluble form of the piwi protein and gene therapy approaches using an isolated and purified polynucleotide of the present invention.
Thus, a key aspect of this invention pertains to the discovery of the novel piwi family of proteins and nucleic acids. Preferred nucleic acid and amino acid sequences are described in SEQ ID NOs:1-6.
It is thus another aspect of this invention to provide a purified and isolated piwi family polypeptide having a role in the growth, proliferation and/or self-renewing division of stem cells, and proliferation of primordial germ cells.
The foregoing aspects and embodiments have broad utility given the biological significance of stem cell growth, proliferation and renewal, and primordial germ cell proliferation. By way of example, the foregoing aspects and embodiments are useful in the preparation of screening assays and assay kits that are used to identify compounds that affect or modulate piwi family biological activity, or that are used to detect the presence of the proteins and nucleic acids of this invention in biological samples. Additionally, it is well known that isolated and purified polypeptides have utility as feed additives for livestock and further polynucleotides encoding the polypeptides are thus useful in producing the polypeptides.
Some of the aspects and objects of the invention having been stated hereinabove, other aspects and objects will become evident as the description proceeds, when taken in connection with the accompanying Examples and Drawings as best described hereinbelow.